TWI750905B

TWI750905B - Thiazole compounds as protein kinase inhibitors

Info

Publication number: TWI750905B
Application number: TW109140481A
Authority: TW
Inventors: 蔣維棠; 趙宇生
Original assignee: 財團法人國家衛生研究院; 泰緯生命科技股份有限公司
Priority date: 2020-11-19
Filing date: 2020-11-19
Publication date: 2021-12-21
Also published as: TW202220982A

Abstract

Thiazole compounds of Formula (I) shown below and pharmaceutical compositions containing one of such compounds:

wherein R₁ and R₂ are defined in the specification. Also disclosed are methods of inhibiting a tyrosine kinase and treating cancer associated with a tyrosine kinase with one of the thiazole compounds.

Description

噻唑化合物作為蛋白質激酶抑制劑 Thiazole compounds as protein kinase inhibitors

本發明提供一種噻唑化合物及包含其之醫藥組成物，可用於治療與酪胺酸激酶相關的癌症。 The present invention provides a thiazole compound and a pharmaceutical composition containing the same, which can be used to treat cancer related to tyrosine kinase.

蛋白激酶在調節各種細胞功能的細胞訊號路徑是重要的，包含分化、增生、遷移和凋亡。蛋白激酶的失調涉及癌症和許多其他的疾病。 Protein kinases are important in cellular signaling pathways that regulate various cellular functions, including differentiation, proliferation, migration, and apoptosis. Dysregulation of protein kinases is implicated in cancer and many other diseases.

酪胺酸激酶(tyrosine kinase)為蛋白激酶的一種亞類，透過將磷酸根從ATP中轉移至目標蛋白酪胺酸的羥基基團來調節目標蛋白功能。類FMS酪胺酸激酶3(FMS-like tyrosine kinase 3，FLT3)和酪胺酸激酶Kit(tyrosine-protein kinase Kit，c-Kit)為酪胺酸激酶，已經被研究作為癌症治療中令人注意的治療標靶。 Tyrosine kinases are a subclass of protein kinases that regulate target protein function by transferring phosphate from ATP to the hydroxyl group of target protein tyrosine. FMS-like tyrosine kinase 3 (FLT3) and tyrosine-protein kinase Kit (c-Kit) are tyrosine kinases that have been studied as interesting cancer treatments therapeutic target.

FLT3為一種受體酪胺酸激酶，其突變可導致癌症的發展，例如急性骨髓性白血病(acute myeloid leukemia)。參見Pratz et al.,Current Drug Targets,2010,11(7),781-9。 FLT3 is a receptor tyrosine kinase whose mutations can lead to the development of cancers such as acute myeloid leukemia. See Pratz et al., Current Drug Targets, 2010, 11(7), 781-9.

c-Kit也是一種受體酪胺酸激酶，其參與細胞內訊號傳遞(intracellular signaling)。c-Kit的突變形式在某些癌症的發生中扮演關鍵角色。已證實抑制c-Kit可以有效的治療胃腸道基質腫瘤(gastrointestinal stromal tumor)、急性骨髓性白血病、和黑色素瘤。參見Babaei et al.,Drug Des Devel Ther.,2016 10,2443-2459。 c-Kit is also a receptor tyrosine kinase involved in intracellular signaling. Mutated forms of c-Kit play a key role in the development of certain cancers. already It has been demonstrated that inhibition of c-Kit can effectively treat gastrointestinal stromal tumors, acute myeloid leukemia, and melanoma. See Babaei et al., Drug Des Devel Ther., 2016 10, 2443-2459.

噻唑化合物被廣泛地探索作為有效的酪胺酸激酶抑制劑，其作為候選藥物存在一些挑戰。他們具有較差的激酶選擇性，通常在毒性研究中造成動物死亡，且通常缺乏足夠的體內暴露(in vivo exposure)以在臨床前或臨床研究中發揮期望的功效。 Thiazole compounds are widely explored as potent tyrosine kinase inhibitors, which present some challenges as drug candidates. They have poor kinase selectivity, often kill animals in toxicity studies, and often lack sufficient in vivo exposure to exert desired efficacy in preclinical or clinical studies.

有需要開發新的噻唑化合物，其專一性地抑制特定酪胺酸激酶，展現出期望的安全性，並且在治療目標癌症中發揮足夠的體內功效。 There is a need to develop new thiazole compounds that specifically inhibit specific tyrosine kinases, exhibit the desired safety profile, and exhibit sufficient in vivo efficacy in the treatment of target cancers.

本發明意外地發現特定噻唑化合物可有效地抑制多種酪胺酸激酶，例如類FMS酪胺酸激酶3(FTL3)和酪胺酸激酶Kit(c-Kit)。 The present inventors have unexpectedly discovered that certain thiazole compounds can effectively inhibit a variety of tyrosine kinases, such as FMS-like tyrosine kinase 3 (FTL3) and tyrosine kinase Kit (c-Kit).

在一態樣中，本發明涉及式(I)之噻唑化合物： In one aspect, the present invention relates to thiazole compounds of formula (I):

其中，R₁為C_1-6烷基；以及R₂為雜芳基。 wherein R ₁ is C _1-6 alkyl; and R ₂ is heteroaryl.

在此，術語「烷基」是指含有1-6個碳原子(例如C₁-C₄、C₁-C₃和C₁-C₂)之直鏈或支鏈的烴基團。例子包含甲基、乙基、正丙基、異丙基、正丁基、異丁基和叔丁基。 Here, the term "alkyl" refers to a straight hydrocarbon group containing 1 to 6 carbon atoms (e.g., _{_{_{C 1 -C 4, C 1 -C}}} 3 and C ₁ -C ₂₎ of the chain, or branched. Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and tert-butyl.

術語「雜芳基」是指具有一或多個雜原子(例如O、N、P、和S)的芳香族5-8員單環、8-12員雙環、或11-14員三環系統。例子包含苯硫基(thiophenyl)、***基(triazolyl)、噁唑基(oxazolyl)、噻二唑基(thiadiazolyl)、四唑基(tetrazolyl)、吡唑基(pyrazolyl)、吡啶基(pyridyl)、呋喃基(furyl)、咪唑基(imidazolyl)、苯並咪唑基(benzimidazolyl)、嘧啶基(pyrimidinyl)、噻吩基(thienyl)、喹啉基(quinolinyl)、吲哚基(indolyl)、噻唑基(thiazolyl)和苯並噻唑基(benzothiazolyl)。 The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having one or more heteroatoms (eg, O, N, P, and S) . Examples include thiophenyl, triazolyl, oxazolyl, thiadiazolyl, tetrazolyl, pyrazolyl, pyridyl , furyl, imidazolyl, benzimidazolyl, pyrimidinyl, thienyl, quinolinyl, indolyl, thiazolyl ( thiazolyl) and benzothiazolyl.

除非另有說明，否則本文提及之烷基和雜芳基包含經取代和未經取代之官能基。雜芳基可能的取代基包含C_1-10烷基、C_2-10烯基、C_2-10炔基、C_3-20環烷基、C_3-20環烯基、C_1-20雜環烷基、C_1-20雜環烯基、C_1-10烷氧基、芳基、芳氧基、雜芳基、雜芳氧基、胺基、C_1-10烷胺基、C_1-20二烷胺基、芳胺基、二芳胺基、羥基、鹵素、硫基、C_1-10烷硫基、芳硫基、C_1-10烷磺醯基(alkylsulfonyl)、芳磺醯基(arylsulfonyl)、醯胺基(acylamino)、胺醯基(aminoacyl)、胺基硫代醯基(aminothioacyl)、甲脒基(amidino)、胍基(guanidine)、脲基(ureido)、氰基、硝基、醯基(acyl)、硫醯基(thioacyl)、醯氧基(acyloxy)、羧基(carboxyl)、和羧酸酯基(carboxylic ester)。另一方面，烷基可能的取代基包含上述除了C_1-10烷基、C_2-10烯基和C_2-10炔基以外的所有取代基。環烷基、雜環烷基、芳基和雜芳基也可以彼此稠合。 Unless otherwise indicated, references to alkyl and heteroaryl groups herein include substituted and unsubstituted functional groups. Possible substituents for heteroaryl include C _1-10 alkyl, C _2-10 alkenyl, C _2-10 alkynyl, C _3-20 cycloalkyl, C _3-20 cycloalkenyl, C _1-20 hetero Cycloalkyl, C _1-20 heterocycloalkenyl, C _1-10 alkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, amino, C _1-10 alkylamino, C _{1 -20} dialkylamine, arylamine, diarylamine, hydroxyl, halogen, thio, C _1-10 alkylthio, arylthio, C _1-10 alkylsulfonyl, aromatic sulfonyl arylsulfonyl, acylamino, aminoacyl, aminothioacyl, amidino, guanidine, ureido, cyano , nitro, acyl, thioacyl, acyloxy, carboxyl, and carboxylic ester. On the other hand, the possible substituents of the alkyl group include all the substituents mentioned above except the _C1-10 alkyl group, the _C2-10 alkenyl group and the _C2-10 alkynyl group. Cycloalkyl, heterocycloalkyl, aryl and heteroaryl groups can also be fused to each other.

如果適用，上述噻唑化合物包含化合物本身，以及他們的鹽類、前驅藥(prodrug)、和溶劑合物(solvate)。例如，可以在噻唑化合物上的陰離子和帶正電的基團(如氨基)間形成鹽類，合適的陰離子包含氯、溴、碘、硫酸鹽、硝酸鹽、磷酸鹽、檸檬酸鹽(citrate)、甲磺酸鹽(methanesulfonate)、三氟醋酸鹽 (trifluoroacetate)、醋酸鹽、蘋果酸鹽(malate)、甲苯磺酸鹽(tosylate)、酒石酸鹽(tartrate)、延胡索酸鹽(fumurate)、麩胺酸鹽(glutamate)、葡萄糖醛酸鹽(glucuronate)、乳酸鹽(lactate)、戊二酸鹽(glutarate)和順丁烯二酸鹽(maleate)。同樣地，也可以在噻唑化合物上的陽離子和帶負電的基團(如羧酸鹽)間形成鹽類，合適的陽離子包含鈉離子、鉀離子、鎂離子、鈣離子和銨陽離子例如四甲基銨離子。噻唑化合物也包含那些含有四級氮原子的鹽類。前驅藥的例子包含酯類和其他藥學上可接受的衍生物，其在給予主體時，能夠提供活性的噻唑化合物。溶劑合物是指在活性的噻唑化合物和藥學上可接受的溶劑之間形成的複合物，藥學上可接受的溶劑的例子包含水、乙醇、異丙醇、乙酸乙酯、乙酸和乙醇胺(ethanolamine)。 The aforementioned thiazole compounds include the compounds themselves, as well as their salts, prodrugs, and solvates, as applicable. For example, salts can be formed between an anion on a thiazole compound and a positively charged group such as an amino group, suitable anions include chlorine, bromine, iodine, sulfate, nitrate, phosphate, citrate , methanesulfonate (methanesulfonate), trifluoroacetate (trifluoroacetate), acetate, malate (malate), tosylate (tosylate), tartrate (tartrate), fumarate (fumurate), glutamate (glutamate), glucuronate (glucuronate), Lactate, glutarate and maleate. Likewise, salts can also be formed between cations on thiazole compounds and negatively charged groups such as carboxylates, suitable cations include sodium, potassium, magnesium, calcium and ammonium cations such as tetramethyl Ammonium ions. Thiazole compounds also include those salts containing quaternary nitrogen atoms. Examples of prodrugs include esters and other pharmaceutically acceptable derivatives which, when administered to a subject, provide the active thiazole compound. Solvates refer to complexes formed between an active thiazole compound and a pharmaceutically acceptable solvent, examples of which include water, ethanol, isopropanol, ethyl acetate, acetic acid and ethanolamine ).

在另一態樣中，本發明涉及抑制酪胺酸激酶，例如FLT3和c-Kit的方法，該方法包含使酪胺酸激酶與有效劑量的上述一或多種噻唑化合物接觸。 In another aspect, the present invention relates to a method of inhibiting tyrosine kinases, such as FLT3 and c-Kit, the method comprising contacting the tyrosine kinase with an effective amount of one or more thiazole compounds described above.

治療與酪胺酸激酶相關之癌症的方法也在本發明的範圍內，該方法包含向一需求主體施用一有效劑量的一或多種上述式(I)描述的噻唑化合物。 Also within the scope of the present invention are methods of treating tyrosine kinase-associated cancer, the method comprising administering to a subject in need thereof an effective dose of one or more thiazole compounds described in formula (I) above.

與癌症相關的酪胺酸激酶可以是野生型(wild type)或突變體。酪胺酸激酶的例子包含但不限於類FMS酪胺酸激酶3(FLT3)、類FMS酪胺酸激酶4(FLT4)、血管內皮生長因子受體(VEGFR)、血小板源生長因子受體(platelet-derived growth factor receptor，PDGFR)A、血小板源生長因子受體B(PDGFR B)、酪胺酸激酶Kit(c-Kit)、c-Src激酶(SRC)、酪胺酸激酶Lyn(tyrosine-protein kinase Lyn，LYN)A、酪胺酸激酶Lyn B(LYN B)、在轉染期間重排的酪胺酸激酶(rearranged during transfection tyrosine kinase，RET)、淋巴細胞專一性蛋白質酪胺酸激酶(lymphocyte-specific protein tyrosine kinase)、加德納拉希德貓肉瘤病毒致癌基因同源物(Gardner-Rasheed feline sarcoma viral oncogene homolog)、盤狀結構域受體1(discoidin domain receptor 1)、激酶***結構域受體(kinase insert domain receptor)、B淋巴細胞激酶(B lymphocyte kinase)、酪胺酸激酶Yes(tyrosine-protein kinase Yes)、愛柏森鼠類白血病病毒致癌基因同源物1(Abelson murine leukemia viral oncogene homolog 1，ABL1)、酪胺酸激酶Tek(tyrosine-protein kinase Tek)、RET V804L、RET Y791F、FLT3 D835Y、PDGFR A V561D、或ABL1 T315I。 Cancer-associated tyrosine kinases can be wild type or mutant. Examples of tyrosine kinases include, but are not limited to, FMS-like tyrosine kinase 3 (FLT3), FMS-like tyrosine kinase 4 (FLT4), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (platelet -derived growth factor receptor, PDGFR) A, platelet-derived growth factor receptor B (PDGFR B), tyrosine kinase Kit (c-Kit), c-Src kinase (SRC), tyrosine kinase Lyn (tyrosine-protein kinase Lyn, LYN) A, tyrosine kinase Lyn B (LYN B), rearranged during transfection tyrosine kinase (RET), lymphocyte-specific protein tyrosine kinase (lymphocyte -specific protein tyrosine kinase), Garde Gardner-Rasheed feline sarcoma viral oncogene homolog, discoidin domain receptor 1, kinase insert domain receptor, B lymphocyte kinase (B lymphocyte kinase), tyrosine-protein kinase Yes (tyrosine-protein kinase Yes), Abelson murine leukemia viral oncogene homolog 1 (ABL1), tyrosine Kinase Tek (tyrosine-protein kinase Tek), RET V804L, RET Y791F, FLT3 D835Y, PDGFR A V561D, or ABL1 T315I.

在一示例性方法中，式(I)之噻唑化合物用於治療與FLT3或c-Kit相關的癌症。 In an exemplary method, thiazole compounds of formula (I) are used to treat cancer associated with FLT3 or c-Kit.

癌症的例子包含急性骨髓性白血病(acute myeloid leukemia)、綠色白血病(chloroma)、慢性骨髓性白血病(chronic myelogenous leukemia)、急性淋巴細胞性白血病(acute lymphoblastic leukemia)、慢性淋巴細胞白血病(chronic lymphocytic leukemia)、霍奇金病(Hodgkin’s disease)、非霍奇金淋巴瘤(non-Hodgkin’s lymphoma)、B細胞淋巴瘤(B-cell lymphoma)、多發性骨髓瘤(multiple myeloma)、瓦爾登斯特倫巨球蛋白血症(Waldenstrom’s macroglobulinemia)、骨髓增生異常綜合徵(myelodysplastic syndrome)、胰腺癌(pancreatic cancer)、膀胱癌(bladder cancer)、結直腸癌(colorectal cancer)、乳癌(breast cancer)、男性生殖道癌(male genital tract cancer)、腎癌(renal cancer)、肝細胞癌(hepatocellular cancer)、肺癌(lung cancer)、卵巢癌(ovarian cancer)、子宮頸癌(cervical cancer)、子宮癌(uterus cancer)、妊娠滋養細胞疾病(gestational trophoblastic disease)、胃癌(gastric cancer)、膽道癌(bile duct cancer)、膽囊癌(gallbladder cancer)、小腸癌(small intestine cancer)、食管癌(esophageal cancer)、口咽癌(oropharyngeal cancer)、下嚥癌(hypopharyngeal cancer)、眼癌(eye cancer)、神經癌(nerve cancer)、頭頸部癌症(head and neck cancer)、黑色素瘤(melanoma)、漿細胞瘤(plasmacytoma)、內分泌腺腫瘤(endocrine gland neoplasm)、神經內分泌癌(neuroendocrine cancer)、腦腫瘤(brain tumor)、骨癌(bone cancer)和肉瘤(sarcoma)(例如胃腸道基質腫瘤(gastrointerstinal stromal tumor或GIST))。 Examples of cancer include acute myeloid leukemia, chloroma, chronic myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia , Hodgkin's disease, non-Hodgkin's lymphoma, B-cell lymphoma, multiple myeloma, Waldenstrom macrosphere Waldenstrom's macroglobulinemia, myelodysplastic syndrome, pancreatic cancer, bladder cancer, colorectal cancer, breast cancer, male reproductive tract cancer (male genital tract cancer), kidney cancer (renal cancer), hepatocellular cancer (hepatocellular cancer), lung cancer (lung cancer), ovarian cancer (ovarian cancer), cervical cancer (cervical cancer), uterus cancer (uterus cancer), Gestational trophoblastic disease, gastric cancer, bile duct cancer, gallbladder cancer, small intestine cancer, esophageal cancer, Oropharyngeal cancer, hypopharyngeal cancer, eye cancer, nerve cancer, head and neck cancer, melanoma, plasmacytoma ( plasmacytoma, endocrine gland neoplasm, neuroendocrine cancer, brain tumor, bone cancer and sarcoma (e.g. gastrointerstinal stromal tumor or GIST) )).

在一示例性方法中，式(I)之噻唑化合物用於治療急性骨髓性白血病。 In an exemplary method, thiazole compounds of formula (I) are used to treat acute myeloid leukemia.

在本發明範圍內還有包含一或多種上述式(I)之噻唑化合物的醫藥組成物，該醫藥組成物可用於治療癌症。 Also within the scope of the present invention are pharmaceutical compositions comprising one or more thiazole compounds of formula (I) above, which are useful in the treatment of cancer.

本發明還包含一或多種上述式(I)之噻唑化合物用於製備治療癌症的藥物的用途。 The present invention also includes the use of one or more thiazole compounds of formula (I) above for the preparation of a medicament for the treatment of cancer.

術語「治療」是指施用一或多種該噻唑化合物於具有上述疾病(即癌症)、這種疾病的症狀、或朝向這種疾病之傾向的主體，目的是給予治癒效果，例如治癒、緩解、改變、影響、改善或預防上述疾病，其症狀或對其的傾向。術語「有效劑量」是指賦予治療效果所需的活性化合物的量。如本領域技術人員所認識的，有效劑量會依據治療疾病的類型、施用途徑、賦形劑用法、以及與其他治療性療法共同使用的可能性而變化。 The term "treatment" refers to the administration of one or more of the thiazole compounds to a subject having the above-mentioned disease (i.e., cancer), a symptom of such a disease, or a predisposition toward such a disease, with the aim of imparting a curative effect, such as cure, amelioration, modification , affect, ameliorate or prevent the above diseases, their symptoms or predispositions to them. The term "effective dose" refers to the amount of active compound required to impart a therapeutic effect. As will be appreciated by those skilled in the art, the effective dose will vary depending on the type of disease being treated, the route of administration, the use of excipients, and the possibility of co-use with other therapeutic therapies.

為了實施本發明的方法，具有一或多種以上描述的噻唑化合物的組成物可以腸胃外(parenterally)、口服、鼻、直腸、局部、或頰(buccally)給藥。本文所用術語「腸胃外」是指皮下(subcutaneous)、皮內(intracutaneous)、靜脈內(intravenous)、腹膜內(intraperitoneal)、肌肉內(intramuscular)、關節內(intraarticular)、動脈內(intraarterial)、滑囊(腔)內(intrasynovial)、胸骨內 (intrasternal)、鞘內(intrathecal)、疾病部位內(intralesional)、或頭顱內(intracranial)注射，以及任何合適的灌注技術(infusion technique)。 To practice the methods of the present invention, compositions with one or more of the thiazole compounds described above can be administered parenterally, orally, nasally, rectally, topically, or buccally. The term "parenteral" as used herein refers to subcutaneous, intracutaneous, intravenous, intraperitoneal, intramuscular, intraarticular, intraarterial, Intrasynovial, intrasternal intrathecal, intrathecal, intralesional, or intracranial injection, as well as any suitable infusion technique.

無菌注射組成物可以是在無毒的腸胃外可接受的稀釋劑或溶劑中的溶液或懸浮液，例如在1,3-丁二醇中的溶液。可以使用的可接受的載體和溶劑為甘露醇(mannitol)、水、林格氏溶液(Ringer’s solution)、和等滲透壓的氯化鈉溶液。此外，固定油(fixed oils)通常被使用為溶劑或懸浮介質(例如合成的單-或雙-甘油酯(glyceride))。脂肪酸，例如油酸和其甘油酯衍生物可用在注射劑的製備，其為天然藥學上可接受的油，例如橄欖油、蓖麻油(castor oil)，特別是他們的聚氧乙醇化(polyoxyethylated)形式。這些油溶液或懸浮液也可以含有長鏈醇稀釋劑或分散劑、羧甲基纖維素(carboxymethyl cellulose)、或類似的分散劑。其他常用的界面活性劑例如Tweens和Spans，或其他類似的乳化劑或生物可利用增強劑(bioavailability enhancer)，其通常用於製備藥學上可接受的固體、液體或其他劑型，也可被用於配製上。 The sterile injectable composition can be a solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable carriers and solvents that may be employed are mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, fixed oils are conventionally employed as a solvent or suspending medium (eg, synthetic mono- or di-glycerides). Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils such as olive oil, castor oil, especially in their polyoxyethylated versions . These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, carboxymethyl cellulose, or similar dispersants. Other commonly used surfactants such as Tweens and Spans, or other similar emulsifiers or bioavailability enhancers, which are commonly used in the preparation of pharmaceutically acceptable solid, liquid or other dosage forms, may also be used in preparation.

用於口服給藥的組成物可以是任何口服可接受劑型，包含膠囊、錠劑、乳劑以及水性懸浮液、分散液和溶液。在錠劑情況下，常用之載體包含乳糖及玉米澱粉。通常也添加潤滑劑，例如硬脂酸鎂。對於以膠囊形式的口服給藥，有用的稀釋劑包含乳糖及乾燥的玉米澱粉。當以水性懸浮液或乳劑口服給藥時，活性成分可以被懸浮或溶解在與乳化劑或懸浮劑結合的油相中。若有需要，可以添加特定的甜味劑、調味劑或著色劑。 Compositions for oral administration can be in any orally acceptable dosage form, including capsules, lozenges, emulsions, and aqueous suspensions, dispersions, and solutions. In the case of tablets, carriers commonly used include lactose and cornstarch. Lubricants such as magnesium stearate are also often added. For oral administration in capsule form, useful diluents include lactose and dried cornstarch. When administered orally as aqueous suspensions or emulsions, the active ingredient may be suspended or dissolved in an oily phase combined with emulsifying or suspending agents. If desired, certain sweetening, flavoring or coloring agents may be added.

可以根據藥物製劑領域已知的技術製備鼻用噴霧劑(nasal aerosol)或吸入劑(inhalation)組合物。例如，組合物可以製備成在鹽水、使用苯甲醇或其他合適的防腐劑、提高生物可用率的吸收促進劑、碳氟化合物(fluorocarbon)、及/或其他本領域已知的助溶劑或分散劑中的溶液。 Nasal aerosol or inhalation compositions can be prepared according to techniques known in the pharmaceutical formulation arts. For example, the composition can be prepared in brine, using benzyl alcohol or its Solutions of other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and/or other solubilizers or dispersants known in the art.

具有一或多種上述噻唑化合物的組成物也可以栓劑的形式用於直腸給藥。 Compositions with one or more of the aforementioned thiazole compounds may also be used in the form of suppositories for rectal administration.

在意義上來說，醫藥組成物中的載體必須是「可接受的」，其與組成物的活性成分相容(並且較佳為能夠穩定該活形成分)，且對治療的主體無害。一或多種助溶劑可以作為藥物賦形劑，用來輸送活性化合物1,5-二苯基-戊-1,4-二烯-3-酮(1,5-diphenyl-penta-1,4-dien-3-one)。其他載體的例子包含膠態氧化矽(colloidal silicon oxide)、硬脂酸鎂、纖維素、硫酸月桂酯鈉(sodium laucyl sulfate)和D&C Yellow # 10。 A carrier in a pharmaceutical composition must be "acceptable" in the sense that it is compatible with the active ingredient of the composition (and preferably is capable of stabilizing the active ingredient), and is not injurious to the subject being treated. One or more co-solvents can be used as pharmaceutical excipients to deliver the active compound 1,5-diphenyl-penta-1,4-dien-3-one (1,5-diphenyl-penta-1,4- dien-3-one). Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, sodium laucyl sulfate, and D&C Yellow #10.

在下面的描述中闡述了本發明之一或多個實施例的細節。本發明的其他特徵、目的及優點將於說明書和請求範圍中顯而易見。 The details of one or more embodiments of the invention are set forth in the description below. Other features, objects and advantages of the present invention will become apparent from the description and scope of the claims.

圖1顯示實施例5中西方墨點法(Westen Blot)分析的結果。 Figure 1 shows the results of the Westen Blot analysis in Example 5.

詳細公開了式(I)之噻唑化合物： Thiazole compounds of formula (I) are disclosed in detail:

其中，變數R₁和R₂如上述發明內容段落之定義。 where the variables R ₁ and R _{2 are} as defined in the above summary paragraph.

通常，式(I)之化合物的R₁為C_1-6烷基。R₁的一個實施例為甲基。 _{Typically, R 1} of compounds of formula (I) is C _1-6 alkyl. An example of R _{1 is methyl.}

通常，式(I)之化合物的R₂為5-或6-員雜芳基。示例性化合物的R₂為6-員雜芳基，R₂的一個實施例為吡啶基。 Typically, R in the compound of formula (I) ₂ of a 5- or 6-membered heteroaryl. Exemplary compounds R ₂ is 6-membered heteroaryl, R ₂ is a pyridyl embodiment.

一組上述新型噻唑化合物為式(II)之化合物； One group of the above-mentioned novel thiazole compounds is the compound of formula (II);

在一子集中，式(II)之化合物的R₁為甲基。 In one subset, R a compound of formula (II) of is _methyl.

在另一子集中，式(II)之化合物的R₂為6-員雜芳基。 In another subset, R a compound of formula (II) it _is 6-membered heteroaryl.

在又一子集中，式(II)之化合物的R₂為吡啶基。 In yet another subset, R a compound of formula (II) is the _2-pyridyl.

示例性化合物包含但不限於

。 Exemplary compounds include, but are not limited to

.

同樣在本發明內的是含有一或多種式(I)之噻唑化合物的醫藥組成物，其用於治療癌症。 Also within the present invention are pharmaceutical compositions containing one or more thiazole compounds of formula (I) for use in the treatment of cancer.

本發明還包含治療癌症的方法，該方法包含向一需求主體施用一有效劑量的式(I)之化合物。 The present invention also includes a method of treating cancer comprising administering to a subject in need thereof an effective amount of a compound of formula (I).

用於合成式(I)之化合物的合成化學轉化和保護基團方法(保護和去保護)為本領域所熟知的。參見例如R.Larock,Comprehensive Organic Transformations(2^nd Ed.,VCH Publishers 1999)；P.G.M.Wuts and T.W.Greene,Greene’s Protective Groups in Organic Synthesis(4^th Ed.,John Wiley and Sons 2007)；L.Fieser and M.Fieser,Fieser and Fieser’s Reagents for Organic Synthesis(John Wiley and Sons 1994)；L.Paquette,ed.,Encyclopedia of Reagents for Organic Synthesis(2^nd ed.,John Wiley and Sons 2009)；and G.J.Yu et al.,J.Med.Chem.2008,51,6044-6054. Synthetic chemical transformations and protecting group methods (protection and deprotection) for the synthesis of compounds of formula (I) are well known in the art. See, eg, R. Larock, Comprehensive Organic Transformations (2 ^nd Ed., VCH Publishers 1999); PGMWuts and TW Greene, Greene's Protective Groups in Organic Synthesis (4 ^th Ed., John Wiley and Sons 2007); L. Fieser and M. Fieser , Fieser and Fieser's Reagents for Organic Synthesis (John Wiley and Sons 1994); L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis (2 ^nd ed., John Wiley and Sons 2009); and GJYu et al., J. Med.Chem. 2008, 51, 6044-6054.

如此製備的式(I)化合物可以生化的測定法初步篩選，例如下文實施例2-3所描述之激酶分析(kinase assays)，或細胞分析(cellular assays)，例如下文實施例4-5所描述之體外抗癌症活性分析，即他們在抑制酪胺酸激酶或抑制表達特定酪胺酸激酶的癌症細胞生長的潛力。隨後可以使用體內測定法評估他們，例如異種移植動物模型分析(xenograft animal model assay)，即他們在哺乳動物中抑制腫瘤生長的活性。所選的化合物可以進一步測定以驗證他們在治療癌症上的功效。例如可以將化合物施用至患有癌症的動物(例如小鼠)，並接著評估其治療效果，例如實施例6中所述動物研究。根據該結果，可以研究和確認適當劑量範圍和給藥途徑。 The compounds of formula (I) thus prepared can be initially screened in biochemical assays, such as the kinase assays described in Examples 2-3 below, or cellular assays, such as those described in Examples 4-5 below In vitro anticancer activity assays, ie their potential to inhibit tyrosine kinases or inhibit the growth of cancer cells expressing specific tyrosine kinases. They can then be assessed using in vivo assays, such as the xenograft animal model assay, for their activity in inhibiting tumor growth in mammals. Selected compounds can be further assayed to verify their efficacy in treating cancer. For example, a compound can be administered to an animal (eg, a mouse) with cancer and then evaluated for its therapeutic effect, eg, the animal study described in Example 6. Based on the results, appropriate dosage ranges and routes of administration can be studied and confirmed.

不需要進一步說明，相信本領域技術人員可基於以上描述最大程度地利用本發明。因此，以下的具體實施例應被解釋為僅僅是說明性的，而不以任何方式限制本公開的其餘部分，本文引用的所有出版物均併入本發明以供參考。 Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. Accordingly, the following specific examples should be construed to be illustrative only and not to limit the remainder of the disclosure in any way, and all publications cited herein are incorporated herein by reference.

以下實施例1所示為式(I)之示例性化合物的合成及特性。如此製備的化合物的分析資料也列於實施例1中，且用於測試化合物的步驟於隨後的實施例2-6中描述。 Example 1 below shows the synthesis and characterization of exemplary compounds of formula (I). Analytical data for the compounds so prepared are also presented in Example 1, and the procedures used to test the compounds are described in Examples 2-6 that follow.

所有化學藥品和溶劑從商業供應商購買，並以原樣使用。所有反應在乾燥氮氣氣氛下進行。使用Merk 60 F254矽膠玻璃背板(5 x 10cm)的薄層層析法(TLC)監測反應，且在紫外線(254nm)照射下或藉由以磷鉬酸試劑(Aldrich)噴霧後，接著在80℃下加熱目視檢測區域。所有急速管柱層析使用Merck Kieselgel 60,No.9385,230-400網目(mesh)ASTM矽膠作為固定相進行。質子(¹H)核磁共振光譜以Varian Mercury-300和Varian Mercury-400光譜儀測量。以相對於溶劑峰的共振在δ尺度上以百萬分之一(ppm)紀錄化學位移。以下縮寫用於描述耦合：s=單峰；d=雙重峰；t=三重峰；q=四重峰；quin=五重峰；br=寬；以及m=多重峰。液相色譜-質譜(LCMS)數據以Agilent MSD-1100 ESI-MS/MS,Agilent 1200 series LC/MSD VL和Waters Acquity UPLC-ESI-MS/MS系統測量。 All chemicals and solvents were purchased from commercial suppliers and used as received. All reactions were carried out under a dry nitrogen atmosphere. The reaction was monitored by thin-layer chromatography (TLC) using a Merk 60 F254 silica glass backplane (5 x 10 cm), and after irradiation with UV light (254 nm) or by spraying with phosphomolybdic acid reagent (Aldrich), then at 80 Heat the visual inspection area at °C. All flash column chromatography was performed using Merck Kieselgel 60, No. 9385, 230-400 mesh ASTM silica gel as the stationary phase. Proton ( ¹ H) nuclear magnetic resonance spectra were measured with Varian Mercury-300 and Varian Mercury-400 spectrometers. Chemical shifts are reported in parts per million (ppm) on the delta scale as resonances relative to the solvent peaks. The following abbreviations are used to describe coupling: s=singlet; d=doublet; t=triplet; q=quartet; quin=quintet; br=broad; and m=multiplet. Liquid chromatography-mass spectrometry (LCMS) data were measured on Agilent MSD-1100 ESI-MS/MS, Agilent 1200 series LC/MSD VL and Waters Acquity UPLC-ESI-MS/MS systems.

實施例1：化合物1之合成 Example 1: Synthesis of Compound 1

根據以下程序1所示的合成路徑製備化合物1。在所列的試劑中，DMSO為二甲基亞碸，以及HCl為鹽酸。根據已公開的步驟(J.Med.Chem.2019,62,11135)製備起始物A。 Compound 1 was prepared according to the synthetic route shown in Scheme 1 below. Among the listed reagents, DMSO is dimethylsulfoxide, and HCl is hydrochloric acid. Starting material A was prepared according to published procedures (J. Med. Chem. 2019, 62, 11135).

[程序1] [Program 1]

試劑和條件：(a)哌嗪(Piperazine),DMSO,70℃；以及(b)6N HCl,0℃ Reagents and conditions: (a) Piperazine, DMSO, 70°C; and (b) 6N HCl, 0°C

將溶於二甲基亞碸(2mL)的化合物A(2mmol)和哌嗪(20mmol)的混合物在70℃下加熱2小時。冷卻至室溫後，將混合物以水稀釋(50mL)。藉由過濾收集沉澱物，以水(10mL)洗並真空乾燥。殘餘物經由氧化鋁色層分析法純化(0.5%至1.5%梯度的甲醇/二氯甲烷)，得到化合物1的游離鹼，其為灰白色(off-white)固體。 A mixture of Compound A (2 mmol) and piperazine (20 mmol) dissolved in dimethylsulfite (2 mL) was heated at 70°C for 2 hours. After cooling to room temperature, the mixture was diluted with water (50 mL). The precipitate was collected by filtration, washed with water (10 mL) and dried in vacuo. The residue was purified via alumina chromatography (0.5% to 1.5% gradient methanol/dichloromethane) to give the free base of compound 1 as an off-white solid.

在0℃下向攪拌的6N鹽酸(2mL)中添加上述固體，並將溶液通過0.45μm聚偏二氟乙烯(PVDF)膜過濾。在1小時內向攪拌的濾液中逐滴添加丙酮 (20mL)，並在0℃下再攪拌1小時。藉由過濾收集沉澱物，以丙酮(10mL)洗並真空乾燥，以得到黃色固體的化合物1的鹽酸鹽(85%)。 To stirring 6N hydrochloric acid (2 mL) was added the above solid at 0°C, and the solution was filtered through a 0.45 μm polyvinylidene fluoride (PVDF) membrane. Acetone was added dropwise to the stirred filtrate over 1 hour (20 mL) and stirred for an additional 1 h at 0 °C. The precipitate was collected by filtration, washed with acetone (10 mL) and dried in vacuo to give the hydrochloride salt of Compound 1 (85%) as a yellow solid.

2-甲基-6-(哌嗪-1-基)-N-[5-(吡啶-4-基)-1,3-噻唑-2-基]嘧啶-4-胺二鹽酸鹽 (2-Methyl-6-(piperazin-1-yl)-N-[5-(pyridine-4-yl)-1,3-thiazol-2-yl]pyrimidin-4-amine dihydrochloride)(化合物1)。¹H NMR(400MHz,D₂O)：δ8.65(d,2H,J=7.2Hz),8.32(s,1H),8.11(d,J=7.2Hz,2H),6.41(s,1H),4.08(t,J=5.4Hz,4H),3.45(t,J=5.2Hz,4H),2.66(s,3H)；¹³C NMR(100MHz,D₂O)：δ 164.3,161.4,159.3,153.9,148.5,141.3,140.8,125.9,121.8,84.7,42.5,42.1,21.8；MS(ES⁺)m/z理論值C₁₇H₁₉N₇S：353.1；實際值：354.1[M+H]⁺. 2-Methyl-6-(piperazin-1-yl)-N-[5-(pyridin-4-yl)-1,3-thiazol-2-yl]pyrimidin-4-amine dihydrochloride (2 -Methyl-6-(piperazin-1-yl)-N-[5-(pyridine-4-yl)-1,3-thiazol-2-yl]pyrimidin-4-amine dihydrochloride) (compound 1). ¹ H NMR (400 MHz, D ₂ O): δ 8.65 (d, 2H, J = 7.2 Hz), 8.32 (s, 1 H), 8.11 (d, J =7.2 Hz, 2H), 6.41 (s, 1 H) , 4.08(t, J =5.4Hz, 4H), 3.45(t, J =5.2Hz, 4H), 2.66(s, 3H); ¹³ C NMR (100 MHz, D ₂ O): δ 164.3, 161.4, 159.3, 153.9, 148.5, 141.3, 140.8, 125.9, 121.8, 84.7, 42.5, 42.1, 21.8; MS(ES ⁺ ) m/z theoretical value C ₁₇ H ₁₉ N ₇ S: 353.1; actual value: 354.1 [M+H] ⁺ .

實施例2：FLT3活性抑制 Example 2: FLT3 activity inhibition

進行以下研究以測試根據實施例1製備的化合物1鹽酸鹽對FLT3活性抑制。 The following studies were performed to test the inhibition of FLT3 activity by Compound 1 hydrochloride prepared according to Example 1.

含有FLT3激酶催化域(catalytic domain)(殘基Y567-S993)的GST-FLT3-KD^WT在轉染(transfected)含有pBac-PAK8-GST-FLT3-KD質體(plasmid)的桿狀病毒(baculovirus)的Sf9昆蟲細胞(insect cells)中表達。FLT3^WT Kinase-Glo測定在30℃下於96孔盤中進行4小時以測試化合物，最終體積為50μL的化合物包含以下成分：75ng GST-FLT3-KD^WT蛋白、25mM HEPES，pH 7.4、4mM氯化錳(MnCl₂)、10mM氯化鎂(MgCl₂)、2mM DTT、0.02% Triton X-100、0.1mg/mL牛血清蛋白(bovine serum albumin)、25μM Her2胜肽基質(peptide substrate)、0.5mM正釩酸鈉(Na₃VO₄)和1μM ATP。培育後，將50μL的Kinase-Glo Plus試劑(Promega,Madison,WI,USA)添加至每個孔中，並將混合物在25℃下培育20分鐘。將70μL等分的每個反應混合物轉移至黑色微量培養盤(microtiter plate)中，並以Wallac Vector 1420多標記計數器(multilabel counter)(PerkinElmer,Shelton,CT,USA)量測螢光。 ^{GST-FLT3-KD WT} containing FLT3 kinase catalytic domain (residues Y567-S993) was transfected with baculovirus containing pBac-PAK8-GST-FLT3-KD plastid ) in Sf9 insect cells. The FLT3 ^WT Kinase-Glo assay was performed in 96-well plates for 4 hours at 30°C to test compounds, and a final volume of 50 μL of compound contained the following components: 75ng GST-FLT3-KD ^WT protein, 25mM HEPES, pH 7.4, 4mM Chloride Manganese (MnCl ₂ ), 10 mM magnesium chloride (MgCl ₂ ), 2 mM DTT, 0.02% Triton X-100, 0.1 mg/mL bovine serum albumin, 25 μM Her2 peptide substrate, 0.5 mM vanadium sodium (Na ₃ VO ₄₎ and 1μM ATP. After incubation, 50 μL of Kinase-Glo Plus reagent (Promega, Madison, WI, USA) was added to each well and the mixture was incubated at 25°C for 20 minutes. A 70 μL aliquot of each reaction mixture was transferred to a black microtiter plate and fluorescence was measured with a Wallac Vector 1420 multilabel counter (PerkinElmer, Shelton, CT, USA).

化合物1的IC₅₀(響應降低一半時的抑制劑濃度)值低於100nM。 1 Compound IC ₅₀ (concentration of inhibitor response to a decrease in half) value of less than 100nM.

實施例3：c-Kit活性抑制 Example 3: c-Kit activity inhibition

進行以下研究以測試根據實施例1製備的化合物1鹽酸鹽對c-Kit活性抑制。 The following study was conducted to test the inhibition of c-Kit activity by Compound 1 hydrochloride prepared according to Example 1.

純化在Sf9桿狀病毒-昆蟲細胞表達系統中表達的N-末端His-標記的(His-tagged)人類c-Kit(殘基T544-V976)重組蛋白(recombinant proteins)，以用於c-KIT ADP Kinase-Glo測定。c-Kit-ADP Kinase-Glo測定在30℃下於96孔盤中進行150分鐘，最終體積為10μL，包含：40mM Tris pH 7.4、20mM氯化鎂(MgCl₂)、2mM氯化錳(MnCl₂)、2mM DTT、0.01% BSA、20μM ATP、20μM poly(Glu,Tyr)4：1胜肽、0.1mM正釩酸鈉(Na₃VO₄)、250ng的重組c-Kit蛋白、以及指定濃度的測試化合物。在25℃下培育40分鐘，經由添加5μL ADP-Glo^TM試劑(Promega,Madison,WI,USA)來終止反應，接著添加10μL激酶檢測試劑持續30分鐘。最後，將30μL等分的每個反應混合物轉移至黑色微量培養盤(microtiter plate)，並以Wallac Vector 1420多標記計數器(PerkinElmer,Shelton,CT,USA)量測螢光。 Purification of N-terminal His-tagged human c-Kit (residues T544-V976) recombinant proteins expressed in the Sf9 baculovirus-insect cell expression system for use in c-KIT ADP Kinase-Glo assay. The c-Kit-ADP Kinase-Glo assay was performed at 30°C for 150 min in a 96-well plate in a final volume of 10 μL containing: 40 mM Tris pH 7.4, 20 mM magnesium chloride (MgCl ₂ ), 2 mM manganese chloride (MnCl ₂ ), 2mM DTT, 0.01% BSA, 20μM ATP, 20μM poly (Glu, Tyr) 4: 1 peptide, 0.1 mM sodium orthovanadate (Na ₃ VO _4), c-Kit recombinant protein 250ng, and specified concentrations of test compound . After incubation at 25°C for 40 minutes, the ^{reaction was stopped by the addition of 5 μL of ADP-Glo™} reagent (Promega, Madison, WI, USA), followed by the addition of 10 μL of kinase detection reagent for 30 minutes. Finally, 30 μL aliquots of each reaction mixture were transferred to black microtiter plates and fluorescence was measured with a Wallac Vector 1420 multilabel counter (PerkinElmer, Shelton, CT, USA).

化合物1的IC₅₀值低於100nM。 IC ₅₀ values of compound 1 below 100nM.

細胞株和細胞培養 Cell Lines and Cell Culture

MV4：11、BaF3和HEK293T細胞株自美國典型培養物保藏中心(American Type Culture Collection,ATCC,Manassas,VA,USA)獲得。MOLM-13細胞株自Deutsche Sammlung von Microorganismen und Zellkulturen GmbH (DSMZ,Braunschweig,Germany)購得。GIST-T1細胞株自COSMO BIO CO.,LTD(Tokyo,Japan)獲得。將GIST-T1、MOLM-13、MV4：11、BaF3、BaF3/FLT3-D835Y、BaF3/FLT3-ITD-F691L和BaF3/FLT3-ITD-D835Y細胞在37℃於5%CO₂下，維持在補充有10%胎牛血清(fetal bovine serum,FBS)、10U/mL盤尼西林和100μg/mL鏈黴素的RPMI 1640培養基中。將kasumin-1細胞培養在補充有20%胎牛血清(FBS)、1mM Hepes、1mM丙酮酸鈉(sodium pyruvate)、10U/ml盤尼西林和100μg/ml鏈黴素的RPMI 1640培養基中。將HEK293T和FLT3-轉染的HEK293T細胞培養在含有10% FBS胎牛血清的DMEM (Invitrogen,USA)培養基中。 MV4:11, BaF3 and HEK293T cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). MOLM-13 cell line was purchased from Deutsche Sammlung von Microorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany). GIST-T1 cell line was obtained from COSMO BIO CO., LTD (Tokyo, Japan). The GIST-T1, MOLM-13, MV4: 11, BaF3, BaF3 / FLT3-D835Y, BaF3 / FLT3-ITD-F691L and BaF3 / FLT3-ITD-D835Y cells at 37 [deg.] C in 5% CO _2, are maintained in in RPMI 1640 medium with 10% fetal bovine serum (FBS), 10 U/mL penicillin and 100 μg/mL streptomycin. Kasumin-1 cells were cultured in RPMI 1640 medium supplemented with 20% fetal bovine serum (FBS), 1 mM Hepes, 1 mM sodium pyruvate, 10 U/ml penicillin and 100 μg/ml streptomycin. HEK293T and FLT3-transfected HEK293T cells were cultured in DMEM (Invitrogen, USA) medium containing 10% FBS fetal bovine serum.

GIST882、GIST48和GIST430細胞皆培養在維持37℃和5%CO₂下的培養箱中。GIST882培養在補充有20%胎牛血清(FBS)的RPMI-1640培養基中。GIST48培養在補充有20% FBS、0.5% Mito、血清稀釋液(serum extender,BD Bioscience,355006)和1%牛腦下垂體萃取物(pituitary extract bovine,BD Bioscience 354123)的F10培養基中。GIST430培養在補充有20% FBS的IMDM培養基中。GIST882、GIST430和GIST48細胞由Dr.Jonathan A.Fletcher(Harvard Medical School,US)提供。 GIST882, GIST48 and GIST430 cells were all cultured in an incubator _{maintained at 37°C and 5% CO 2 .} GIST882 was cultured in RPMI-1640 medium supplemented with 20% fetal bovine serum (FBS). GIST48 was cultured in F10 medium supplemented with 20% FBS, 0.5% Mito, serum extender (serum extender, BD Bioscience, 355006) and 1% pituitary extract bovine (BD Bioscience 354123). GIST430 was cultured in IMDM medium supplemented with 20% FBS. GIST882, GIST430 and GIST48 cells were provided by Dr. Jonathan A. Fletcher (Harvard Medical School, US).

體外抗癌活性 In vitro anticancer activity

實施例4：MTS細胞存活率測定 Example 4: MTS cell viability assay

使用細胞株和MTS細胞存活率測定(MTS代表3-(4,5-二甲基噻唑-2-基)-5-(3-羧基甲氧基苯基)-2-(4-磺苯基)-2H-四唑鎓 (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetr azolium))，進行以下研究以評估根據實施例1所製備的測試化合物1鹽酸鹽的體外抗癌活性。 Cell viability assay using cell lines and MTS (MTS stands for 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl) )-2H-tetrazolium (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetr azolium)), the following studies were performed to evaluate the The in vitro anticancer activity of compound 1 hydrochloride was tested.

通過在96孔培養盤中的每孔接種1×10⁴個細胞(MOLM-13、MV4：11、BaF3、BaF3/FLT3-D835Y、BaF3/FLT3-ITD-D835Y和BaF3/FLT3-ITD-F691L)進行細胞存活率測定。GIST-T1細胞以密度8×10³個細胞/100μL接種至96孔培養盤中。16小時後，在培養基中以媒液(vehicle)或各種濃度的化合物處理細胞72小時。根據製造商建議的方案，使用CellTiter 96 AQueous MTS方法(Promega,Madison,WI,USA)量化活細胞。藉由使用培養盤讀取儀(Victor2；PerkinElmer,Shelton,CT,USA)量測490nm的吸光度來判斷結果。IC₅₀定義為與DMSO-處理的(媒液(vehicle))對照相比，造成細胞存活率下降50%的化合物的量，並使用Prism version 6版軟體(GraphPad,San Diego,CA,USA)計算。 Each well by 96 well culture plates were seeded 1 × 10 ⁴ cells (MOLM-13, MV4: 11 , BaF3, BaF3 / FLT3-D835Y, BaF3 / FLT3-ITD-D835Y and BaF3 / FLT3-ITD-F691L) Cell viability assays were performed. GIST-T1 cells were seeded into 96-well culture dishes at a density of 8×10 ^{3 cells/100 μL.} After 16 hours, cells were treated with vehicle or various concentrations of compound in culture medium for 72 hours. Viable cells were quantified using the CellTiter 96 AQueous MTS method (Promega, Madison, WI, USA) according to the manufacturer's suggested protocol. Results were judged by measuring absorbance at 490 nm using a plate reader (Victor2; PerkinElmer, Shelton, CT, USA). IC ₅₀ is defined as the process of comparison with DMSO- (vehicle solution (Vehicle)) control, the cell viability decreased to 50% of the amount of the compound, using Prism version version 6 software (GraphPad, San Diego, CA, USA) is calculated .

以不同劑量的化合物處理GIST細胞(4×10⁴)。在37℃於5%二氧化碳中，將處理過的GIST882細胞培育144小時，並將GIST48和GIST430細胞培育120小時。通過將細胞與亞甲基藍(Clontech,CA,US)培育1小時來測定細胞增殖。使用SpectraMax M5微量培養盤讀取儀(Molecular Devices,US)在450nm處量測吸光度。 Different doses of compound GIST treated cells ⁽⁴ × 10 4). Treated GIST882 cells were incubated for 144 hours and GIST48 and GIST430 cells were incubated for 120 hours at 37°C in 5% carbon dioxide. Cell proliferation was determined by incubating cells with methylene blue (Clontech, CA, US) for 1 hour. Absorbance was measured at 450 nm using a SpectraMax M5 microplate reader (Molecular Devices, US).

式(I)的測試化合物1鹽酸鹽的GI₅₀(最大抑制細胞增殖50%的濃度)值顯示於下表1。 _{The GI 50} (concentration that inhibits maximal cell proliferation by 50%) values of the test compound 1 hydrochloride of formula (I) are shown in Table 1 below.

表1

Table 1

實施例5：西方墨點法(Western Blot Analysis) Example 5: Western Blot Analysis

將轉染的HEK293T細胞溶解於裂解緩衝液(lysis buffer)(50mM Tris、pH 8.0，150mM NaCl、1% Triton X-100、0.5%去氧膽酸鈉(sodium deoxycholate)、0.1% SDS、1mM正釩酸鈉(sodium orthovanadate)、1mM PMSF和1mM DTT)中。蛋白裂解物(lysates)通過SDS-PAGE分離，並轉移至聚偏二氟乙烯(polyvinylidene difluoride,PVDF)膜(Millipore,Bedford,MA,USA)上。用適當的抗體對膜進行免疫轉印(immunoblotted)，並使用SuperSignal試劑(Pierce,Rockford,IL,USA)進行檢測，然後將其暴露於X-ray膠片。購買抗-pFLT3-Tyr591(anti-pFLT3-Tyr591)(#3461,Cell Signaling Technology)抗體用於西方墨點法。 Transfected HEK293T cells were lysed in lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM positive sodium orthovanadate, 1 mM PMSF and 1 mM DTT). Protein lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Membranes were immunoblotted with appropriate antibodies and probed using SuperSignal reagents (Pierce, Rockford, IL, USA) and then exposed to X-ray film. Anti-pFLT3-Tyr591 (anti-pFLT3-Tyr591) (#3461, Cell Signaling Technology) antibody was purchased for Western blotting.

如圖1所示，分析了表達FLT3(FLT3-WT(野生型(wild type))、FLT3-ITD、FLT3-D835Y、FLT3-ITD-D835Y和FLT3-ITD-F691L)的HEK293T細胞。以各種濃度的化合物1鹽酸鹽處理FLT3-轉染的HEK293T細胞1小時。 As shown in Figure 1, HEK293T cells expressing FLT3 (FLT3-WT (wild type), FLT3-ITD, FLT3-D835Y, FLT3-ITD-D835Y and FLT3-ITD-F691L) were analyzed. FLT3-transfected HEK293T cells were treated with various concentrations of Compound 1 hydrochloride for 1 hour.

實施例6：動物研究 Example 6: Animal studies

將化合物1鹽酸鹽配置在20%(2-羥基丙基)-β-環糊精((2-hydroxypropyl)-β-cyclodextrin)(Sigma)。將Kasumi-1細胞(1×10⁶/小鼠)皮下注射至SCID(6至8周齡)雄性小鼠中。當平均腫瘤體積到達約230mm³(n=8)時，將動物隨機分組，然後依照下表所示的劑量和時間表口服化合物1鹽酸鹽。用數位卡尺(digital caliper)量測腫瘤大小，並通過下式計算腫瘤體積(mm³)：體積=(長度x寬度^2)/2。每天監測所有小鼠的毒性跡象。每周量測兩次體重和腫瘤大小。在實驗期間可以每日觀察健康狀況。在研究結束時，動物通過吸入二氧化碳後頸椎脫臼(cervical dislocation)使其安樂死(euthanized)。 Compound 1 hydrochloride was formulated in 20% (2-hydroxypropyl)-β-cyclodextrin (Sigma). The Kasumi-1 cells (1 × 10 ⁶ / mouse) were injected subcutaneously into SCID (6 to 8 weeks old) male mice. When the mean tumor volume reached approximately 230 ^mm3 (n=8), animals were randomized and administered orally Compound 1 hydrochloride according to the doses and schedules shown in the table below. With digital calipers (digital caliper) to measure tumor size and tumor volume was calculated by the following formula (mm ^3): Volume = (length x width ^ 2) / 2. All mice were monitored daily for signs of toxicity. Body weight and tumor size were measured twice a week. Health status can be observed daily during the experiment. At the end of the study, animals were euthanized by cervical dislocation following carbon dioxide inhalation.

下表2顯示了對Kasumi-1異體移植(xenograft)的體內功效研究的結果。 Table 2 below shows the results of an in vivo efficacy study on Kasumi-1 xenograft.

表2

Table 2

以上實施例中顯示的結果表明，化合物1或其鹽類確實具有抑制酪胺酸激酶或抑制表達特定酪胺酸激酶的癌細胞生長的效力。因此，化合物1或其鹽類能夠用於治療與FLT3或c-Kit相關的癌症。 The results shown in the above examples demonstrate that Compound 1 or a salt thereof does have potency to inhibit tyrosine kinase or to inhibit the growth of cancer cells expressing a specific tyrosine kinase. Therefore, Compound 1 or a salt thereof can be used to treat cancers associated with FLT3 or c-Kit.

其他實施例 other embodiments

在本說明書中揭露的所有特徵可以任意組合來組合。本說明書中揭露的每個特徵可以通過具有相同、等同或相似目的的替代特徵來取代。因此，除非另有說明，否則所揭露的每個特徵僅是一系列的等同或相似特徵的一個示例。 All features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent or similar purpose. Thus, unless stated otherwise, each feature disclosed is one example only of a series of equivalent or similar features.

從以上描述中，本領域技術人員可以容易地確定本發明的基本特徵，且在不脫離本發明的精神和範圍的情況下，可以對本發明進行各種改變及修飾以適應各種用途和條件。因此，其他實施例也在所附專利範圍的範圍內。 From the above description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope of the invention, can make various changes and modifications of the invention to adapt it to various usages and conditions. Accordingly, other embodiments are also within the scope of the appended patent.

Claims

一種下式(I)之化合物或其藥學上可接受的鹽：

其中，R₁為C_1-6烷基；以及R₂為雜芳基。 A compound of the following formula (I) or a pharmaceutically acceptable salt thereof:

wherein R ₁ is C _1-6 alkyl; and R ₂ is heteroaryl.

如請求項1所述之化合物或其藥學上可接受的鹽，其中，R₁為甲基。 The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R ₁ is methyl.

如請求項1所述之化合物或其藥學上可接受的鹽，其中，R₂為6員雜芳基。 The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R ₂ is a 6-membered heteroaryl group.

如請求項3所述之化合物或其藥學上可接受的鹽，其中，R₂為吡啶基。 The compound or a pharmaceutically acceptable salt thereof according to claim 3, wherein R ₂ is pyridyl.

如請求項1所述之化合物或其藥學上可接受的鹽，其中，該化合物為式(II)：

The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is of formula (II):

如請求項5所述之化合物或其藥學上可接受的鹽，其中，R₁為甲基。 The compound according to claim 5 or a pharmaceutically acceptable salt thereof, wherein R ₁ is methyl.

如請求項5所述之化合物或其藥學上可接受的鹽，其中，R₂為6員雜芳基。 The compound of claim 5 or a pharmaceutically acceptable salt thereof, wherein R ₂ is a 6-membered heteroaryl group.

如請求項7所述之化合物或其藥學上可接受的鹽，其中，R₂為吡啶基。 The compound of claim 7 or a pharmaceutically acceptable salt thereof, wherein R ₂ is pyridyl.

如請求項1所述之化合物或其藥學上可接受的鹽，其中，該化合物為

The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is

一種醫藥組成物，包含：一藥學上可接受之載體；以及一下式(I)之化合物或其藥學上可接受的鹽：

其中，R₁為C_1-6烷基；以及R₂為雜芳基。 A pharmaceutical composition, comprising: a pharmaceutically acceptable carrier; and a compound of the following formula (I) or a pharmaceutically acceptable salt thereof:

wherein R ₁ is C _1-6 alkyl; and R ₂ is heteroaryl.

如請求項10所述之醫藥組成物，其中，該化合物為

The pharmaceutical composition according to claim 10, wherein the compound is

一種下式(I)之化合物或其藥學上可接受的鹽的用途，用以製備抑制酪胺酸激酶的藥物：

其中，R₁為C_1-6烷基；以及R₂為雜芳基。 Use of a compound of the following formula (I) or a pharmaceutically acceptable salt thereof for the preparation of a tyrosine kinase inhibitor:

wherein R ₁ is C _1-6 alkyl; and R ₂ is heteroaryl.

如請求項12所述之用途，其中，該化合物為

The use according to claim 12, wherein the compound is

一種下式(I)之化合物或其藥學上可接受的鹽的用途，用以製備治療與酪胺酸激酶相關之癌症之藥物：

其中，R₁為C_1-6烷基；以及R₂為雜芳基。 Use of a compound of the following formula (I) or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment of cancer associated with tyrosine kinase:

wherein R ₁ is C _1-6 alkyl; and R ₂ is heteroaryl.

如請求項14所述之用途，其中，該酪胺酸激酶為類FMS酪胺酸激酶3(FLT3)、類FMS酪胺酸激酶4、血管內皮生長因子受體(VEGFR)、集落刺激因子1受體(CSF1R)、血小板源生長因子受體(PDGFR)A、血小板源生長因子受體B、酪胺酸激酶Kit(c-KIT)、c-Src激酶(SRC)、酪胺酸激酶Lyn(LYN)A、酪胺酸激酶Lyn B、在轉染期間重排的酪胺酸激酶(RET)、淋巴細胞專一性蛋白質酪胺酸激酶、加德納拉希德貓肉瘤病毒致癌基因同源物、盤狀結構域受體1、激酶***結構域受體、B淋巴細胞激酶、酪胺酸激酶Yes、愛柏森鼠類白血病病毒致癌基因同源物1(ABL1)、酪胺酸受體激酶TRKA、TRKB、TRKC、ZAK/MLTK、IRAK4、RET V804L、RET Y791F、FLT3 D835Y、PDGFR A V561D、或ABL1 T315I。 The use according to claim 14, wherein the tyrosine kinase is FMS-like tyrosine kinase 3 (FLT3), FMS-like tyrosine kinase 4, vascular endothelial growth factor receptor (VEGFR), colony stimulating factor 1 receptor (CSF1R), platelet-derived growth factor receptor (PDGFR) A, platelet-derived growth factor receptor B, tyrosine kinase Kit (c-KIT), c-Src kinase (SRC), tyrosine kinase Lyn ( LYN)A, tyrosine kinase Lyn B, tyrosine kinase rearranged during transfection (RET), lymphocyte-specific protein tyrosine Acid Kinase, Gardner Rashid Feline Sarcoma Virus Oncogene Homolog, Discoid Domain Receptor 1, Kinase Insertion Domain Receptor, B Lymphocyte Kinase, Tyrosine Kinase Yes, Epson Murine Leukemia Virus Oncogene homolog 1 (ABL1), tyrosine receptor kinase TRKA, TRKB, TRKC, ZAK/MLTK, IRAK4, RET V804L, RET Y791F, FLT3 D835Y, PDGFR A V561D, or ABL1 T315I.

如請求項15所述之用途，其中，該酪胺酸激酶為類FMS酪胺酸激酶3(FLT3)或酪胺酸激酶Kit(c-KIT)。 The use according to claim 15, wherein the tyrosine kinase is FMS-like tyrosine kinase 3 (FLT3) or tyrosine kinase Kit (c-KIT).

如請求項16所述之用途，其中，該癌症為急性骨髓性白血病、綠色白血病(chloroma)、慢性骨髓性白血病、急性淋巴細胞性白血病、慢性淋巴細胞白血病、霍奇金病、非霍奇金淋巴瘤、B細胞淋巴瘤、多發性骨髓瘤、瓦爾登斯特倫巨球蛋白血症、骨髓增生異常綜合徵、胰腺癌、膀胱癌、結直腸癌、乳癌、男性生殖道癌、腎癌、肝細胞癌、肺癌、卵巢癌、子宮頸癌、子宮癌、妊娠滋養細胞疾病、胃癌、膽道癌、膽囊癌、小腸癌、食管癌、口咽癌、下嚥癌、眼癌、神經癌、頭頸部癌症、黑色素瘤、漿細胞瘤、內分泌腺腫瘤、神經內分泌癌、腦腫瘤、骨癌或肉瘤。 The use according to claim 16, wherein the cancer is acute myeloid leukemia, chloroma, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's disease Lymphoma, B-cell lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, myelodysplastic syndrome, pancreatic cancer, bladder cancer, colorectal cancer, breast cancer, male genital tract cancer, kidney cancer, Hepatocellular carcinoma, lung cancer, ovarian cancer, cervical cancer, uterine cancer, gestational trophoblastic disease, gastric cancer, biliary tract cancer, gallbladder cancer, small bowel cancer, esophageal cancer, oropharyngeal cancer, hypopharyngeal cancer, eye cancer, nerve cancer, Head and neck cancer, melanoma, plasmacytoma, endocrine gland tumor, neuroendocrine cancer, brain tumor, bone cancer or sarcoma.

如請求項17所述之用途，其中，該癌症為急性骨髓性白血病。 The use according to claim 17, wherein the cancer is acute myeloid leukemia.

如請求項14所述之用途，其中，該化合物為

The use according to claim 14, wherein the compound is

如請求項18所述之用途，其中，該化合物為

The use according to claim 18, wherein the compound is